The incurable neurodegenerative Alzheimer’s disease has long been associated with β-amyloid build-up in the brain. While this has been known, direct evidence supporting the close relationship of Alzheimer’s disease and the role of β-amyloid has been hard to come by, until now. Recent Aβ-immunotherapy trials have shown that removing aggregated β-amyloid from symptomatic patients can slow down the disease.

β-amyloid removal slows the progression of Alzheimer’s disease

This breakthrough holds many implications for future treatment and handling of Alzheimer’s disease. While the new evidence is far from being a cure itself, it presents the opportunity for long-term prevention and potential immunoprevention towards the disease. This is in part due to how early the signs and abnormal β-amyloid build-up can begin in Alzheimer’s patients, as well as how complex the disease itself can become. However many clinical trials and experiments it may take, groups like LifeTein will always be ready to help supply researchers with the materials they need to make this future possible.

Jucker, M., & Walker, L. C. (2023). Alzheimer’s disease: From immunotherapy to immunoprevention. In Cell (Vol. 186, Issue 20, pp. 4260–4270). Elsevier BV. https://doi.org/10.1016/j.cell.2023.08.021

Please view this video on how to design cell penetrating peptides. The transcript is listed below.

Transcript

Slide 1:

Thank you for joining me. My topic today is the cell-penetrating peptides. My main focus will be the peptide design, peptide synthesis, and its applications.

Slide 2:

First, let me briefly introduce LifeTein. LifeTein was founded in 2008. We have been in the peptide industry for more than ten years. We specialize in peptide synthesis, chemical synthesis, antibody production, and protein services.

Slide 3:

Our main focus is peptide synthesis service. However, over the years, we have expanded to other protein-related areas like protein, antibody services, and products.

Slide 4:

So let us quickly get into the topic: cell-penetrating peptides. What is a cell-penetrating peptide or cpp? From the definition, CPP is a short peptide. They can be about 4-40 aa. The short peptide can enter the cell membrane. They can deliver bioactive cargoes.

Slide 5:

CPPs can also be used to deliver bioactive cargos like siRNAs, DNA, polypeptides, liposomes, nanoparticles, and others, in cells for therapeutic or experimental purposes.

Slide 6:

There are a few popular models for CPP’s entry. 1. The inverted micelle model. The CPPs are positively charged. They interact with the negatively charged phospholipids in the membrane. 2. Direct entry or direct translocation. For example, sequences with multiple Arginines can cause a short-time membrane cytolysis and enter the cells directly. 3. By the traditional method of endocytosis. I will not talk about the details.

Slide 7:

Here are a few examples of the CPP. The most famous examples are HIV tat sequence. The TAT peptide is arginine-rich and can directly penetrate the plasma membrane and stabilize DNA.

Another example is the arginine-rich peptide R8 or R9. We can add stearic acid to the N-terminus.  Stearic acid is a saturated fatty acid with an 18-carbon chain. If you would like to do live cell imaging, we can add fluorescent dye such as Fitc, Alexa fluor, or Cy dye at the N-terminus or C-terminus. I will get to the details.

Slide 8:

CPPs can enter the regular cell membranes. Some other peptides are tissue-targeting peptides. For example, this brain-homing peptide can cross the blood-brain barrier. Other peptides can cross the skin as transdermal peptides, target heart tissues as cardiac targeting peptides, and nuclear localization signal peptides.

Slide 9:

On this slide, I will talk more about the peptide design. There are many ways to make the peptide permeable. In the case of DNA or RNA, you can simply mix the CPPs with oligos. Many transfection reagents are using this mechanism. Simply put, DNA is negatively charged and peptide is positively charged. If you mix them together, they will form small micelles for cell penetration.

However, most of our work is to put the CPPs and your target together by covalent links. For this example, we put eight arginines at the N-terminus of your peptide. A linker called Ahx is added as a spacer. Some users prefer no spacers. It seems that both worked for the purpose. The eight arginines can be put at the C-terminus as well. According to the feedback from our users, most of the N-terminal CPP worked well. A few worked well for the C-terminal conjugation. I guess it depends on the projects.

This example is the Npys linker modification. The cysteine is added to your peptide. We conjugate two sequences together to form a disulfide bond. This is especially useful for the cancer study. Cancer cells have a lower pH of 6.7-7.1. Normal cells have a higher pH of 7.4. Under the acidic environment, the disulfide bond can be cleaved. If your target peptide is a cancer drug candidate, the CPP can introduce the drug cargo to the cancer cell and release the target within the cell. These disulfide-based prodrugs are important for cancer therapy.

If the cysteine is not available for your case, we can add a compound called lysine azide. This method needs click chemistry.

Slide 10:

There are two kinds of click chemistry. The one with copper as the catalyst and the one without. The preferred method is copper-free click chemistry. It is called DBCO and azide reactions. The final conjugate will have a large linker. Many scientists have concerns about the bulky size of the linker. However, some drugs contain bulky linkers without issues or side effects.

Back to Slide 9:

Let us go back to the sequence. The design does not have to be this way. The lysine azide can be any place in the sequence. If you have a head-to-tail cyclic peptide, you can add the lysine azide in the middle. The final product will be like a lollipop, with the CPP as the tail. If the N-terminus is very important to you, you can add the azide at the C-terminus.

Slide 11:

Let us move on to other scenarios. If you would like to track the peptides in live cells, fluorescent dyes can be added. We can do Fitc, Fam, Cy3, cy5, Cy7 and Alexa Fluor. This design will give direct evidence that your target is inside the cells.  

There is a different kind of peptide called peptide nucleic acid or PNA. It is DNA or RNA analog. We can synthesize half as peptide and another half as the PNA.  

This structure is the one I just mentioned earlier. The cyclic tumor targeting RGD peptide can be linked with an R8 cell-penetrating peptide to form a lollipop-shaped structure.

Slide 12:

So far, we have mentioned different ways to conjugate the cargo with cell-penetrating peptides. If your targets are nanoparticles or gold particles. Our requirement is to have active groups like a thiol group or a free amine on it. We have to have the active groups react to the cell-penetrating peptides. It is the same requirement for small compounds.

Slide 13:

The last concept I would like to introduce is the antibody-drug conjugate. This concept is widely accepted in the antibody drug industry. There are three important components: an antibody, a cleavable linker, and the drug.  Once the antibody binds to the target, the drug is released after the hydrolysis by protease.

Slide 14:

The same concept can be used for the peptides. For this concept, we need to screen the best drug candidate for cell entry. The CPP can be tumor-homing peptides, brain-homing peptides, or cardiac targeting peptides I mentioned earlier.

Slide 15:

First, we need to modify the compound. It is better to have a free amine in the compound. Then we can modify the amine group to an azide group. Afterward, we can use the click chemistry for the following conjugation.

Slide 16

LifeTein produced a series of CPPs. They are ready to conjugate your compounds for screening. So far, we have designed and produced more than fifty CPPs.

Slide 17

Step 3 is conjugate peptides with drug candidates.

Slide 18

Once the CPP is conjugated with the drug compound using click chemistry, we can send the final product back to you for further screening. The purpose is to find the best drug delivery system.

Slide 19

To summarize today’s topic, I talked about cell-penetrating peptides with different cargos. As long as you have an active chemical group on the nanoparticles, compounds, or liposomes, we can conjugate the target to any peptide.

Slide 20

That is all for today. Please let me know if you have any questions. Please feel free to contact us by email or phone calls.   

In the world of peptide synthesis, a game-changing innovation has emerged – a remarkable cocktail designed to enhance the cleavage and deprotection of methionine-containing peptides. This groundbreaking concoction, known as Reagent H, is set to transform the landscape of solid-phase peptide synthesis, particularly for those using the 9-fluorenylmethoxycarbonyl (Fmoc) methodology.

Unveiling Reagent H: Your Key to Methionine Side-Chain Protection in Methionine-Containing Peptides

Reagent H, comprised of trifluoroacetic acid (81%), phenol (5%), thioanisole (5%), 1,2-ethanedithiol (2.5%), water (3%), dimethylsulphide (2%), and ammonium iodide (1.5% w/w), has been meticulously crafted to minimize the pesky oxidation of methionine side chains during synthesis. Its exceptional performance is exemplified in the synthesis of a model pentadecapeptide from the active site of DsbC, a pivotal player in protein disulfide bond formation.

The Triumph of Reagent H: Methionine Sulphoxide Conquered

When put to the test, Reagent H outshone its competitors, cocktails K, R, and B. The crude peptides obtained from these widely used mixtures contained a staggering 15% to 55% of methionine sulphoxide. However, Reagent H demonstrated its prowess by yielding pristine peptides devoid of methionine sulphoxide. Remarkably, even when 1.5% w/w NH4I was added to cocktails K, R, and B, they couldn’t match the perfection achieved by Reagent H, although their yield of the desired peptide fell short.

Unraveling the Mysteries: A Closer Look at Methionine-Containing Peptides

But how does Reagent H achieve this remarkable feat? We delve into the proposed mechanism behind its in situ oxidation of cysteine, shedding light on its impressive ability to safeguard methionine side chains while delivering high-quality peptides.

In the world of peptide synthesis, Reagent H stands as a beacon of hope for researchers seeking purity, precision, and protection in their work. Its ability to minimize methionine side-chain oxidation is nothing short of revolutionary, promising a brighter and more efficient future for peptide synthesis enthusiasts. Say goodbye to impurities and hello to perfection with Reagent H.

Many sensory declines accompany aging, one of which is sight. That being said, there is a need for much more research on the visual changes associated with such aging. Specifically, the changes in rod bipolar cells and their ribbon synapses due to aging are an area of interest, along with the complex calcium systems at work. Using a zebrafish model, peptides help determine the effects of aging on vision and the retina.

Zebrafish were used for this experiment thanks to their unique roles as model organisms; they share 70% genomic similarity with humans, and their short lifespan offers the chance to study life cycles in a few short years while still comparable to human aging over decades. Researchers compared data between middle-aged (MA, 18-months-old) and older-aged (OA, 36-months-old) zebrafish, equating to human ages of approximately 38 and 75 years of age, respectively. Using TAMRA ribbon-binding peptides from LifeTein, the team was able to observe changes between the two ages of zebrafish.

What was discovered was a decreased number of synaptic ribbons and increased ribbon length in the OA models. Further, there were many alterations to the local calcium dynamics of the system, implying a more complex change to vision deterioration than initially expected. The model shows how subtle changes could have vast implications for disease models where these alterations may be amplified and surely sheds more light on how human vision may decline with age.

Abhishek P Shrestha, Nirujan Rameshkumar, Johane Martins Boff, Rhea Rajmanna, Thadshayini Chandrasegaran, Frederick E Courtney, David Zenisek, Thirumalini Vaithianathan
bioRxiv 2023.09.01.555825; doi: https://doi.org/10.1101/2023.09.01.555825

LifeTein’s Innovative Peptide Antagonist: A New Ally in the Fight Against Breast Cancer Metastasis

Breast cancer stands as the most commonly diagnosed cancer in women worldwide and is a leading cause of cancer-related deaths. Among its subtypes, triple-negative breast cancer (TNBC) is particularly notorious for being aggressive and prone to metastasizing to vital organs like the brain, lungs, bones, and liver. Despite being more responsive to chemotherapy, TNBC’s propensity for metastasis poses a significant challenge in cancer treatment.

Recent studies, including notable research from the Medical University of Lodz in Poland, have identified a key factor in TNBC metastasis: increased F11R/JAM-A activity. This protein plays a crucial role in the early stages of cancer cell migration across blood vessels, a precursor to metastasis. Enter LifeTein, a pioneering force in peptide technology, which has made a groundbreaking contribution to this research area.

LifeTein provided a specialized peptide antagonist, named P4D, designed to specifically target and inhibit F11R/JAM‑A. The effectiveness of P4D was rigorously tested in lab models. Remarkably, this antagonist not only curbed the proliferation of TNBC cells but also significantly reduced their survival by directly targeting F11R/JAM-A. The result was a notable hindrance in the metastasis process in the mouse models used for the study.

This breakthrough has significant implications. The success of P4D in these preliminary studies suggests potential for future clinical trials and paves the way for more targeted, effective treatments for TNBC, possibly extending to the development of tailored antibodies. LifeTein’s contribution to this field exemplifies its commitment to advancing cancer therapy, offering new hope to those battling with TNBC.

For more detailed insights, refer to the original study by Bednarek, R., Wojkowska, D.W., Braun, M. et al., titled “Triple negative breast cancer metastasis is hindered by a peptide antagonist of F11R/JAM‑A protein,” published in Cancer Cell International.

Bednarek, R., Wojkowska, D.W., Braun, M. et al. Triple negative breast cancer metastasis is hindered by a peptide antagonist of F11R/JAM‑A protein. Cancer Cell Int 23, 160 (2023).

As methods of medicine advance, targeted drug delivery becomes a more appealing and achievable option over its non-selective counterpart. It can focus solely on increasing therapeutic concentration in the target area while greatly eliminating any exposure to healthy tissue, and thus drastically lowering side effects as well. The effective and simple mechanisms of click chemistry are a great way to design payloads for these targeted drug delivery methods. With the use of enzyme-degradable peptides in click chemistry drug delivery, lasting therapeutics can remain in the system for local sustained release over time as well.

Enzyme-degradable peptides for sustained drug delivery

The team at Rutgers focused on a two-phase method to set up the targeted drug delivery. First, ROS-sensitive PEGDA and acrylate-PEG-azide are aimed at the target area, driven by elevated free radical levels. Once the pretargeting is complete, a payload tethered to DBCO is delivered and captured via azide-DBCO reactions. Enzyme-degradable peptides were provided by LifeTein and incorporated into both steps for the ongoing release of the captured payloads.

The results showed success in the models tested, with the initial dosage still effective in capturing the payload several days later. This system demonstrated the versatility of a two-phase method, where long-term effects are even further avoided by incorporating enzyme-degradable peptides. The proof of concept displayed here has great promise for the future of drug delivery and just goes to show how applicable click chemistry is to even more fields.

Emily T. DiMartini, Kelly Kyker-Snowman & David I. Shreiber (2023) A click chemistry-based, free radical-initiated delivery system for the capture and release of payloads, Drug Delivery, 30:1, DOI: 10.1080/10717544.2023.2232952

During cell division, microtubules in the chromosome attach to a region called the centromere. While most species have a single size-restricted centromere, or a monocentromere, some species exist with multiple centromeres distributed across the chromosome, called holocentromeres. What is even more interesting is how holocentric chromosomes are considered to have evolved from the monocentric organisms, and this transition occurred independently across distant lineages, such as green algae, protozoans, invertebrates, as well as flowering plant families. One group aimed to study these holocentromeres more via the lilioid Chionographis japonica. Their goal was to better understand the convergent evolution of holocentromeres studied with peptides.

Peptides help explore holocentromeres

The group determined that the chromosomal localization of the target centromere is usually marked with histone H3 (CENH3). With this knowledge, they utilized peptides and antibodies of CENH3 provided by LifeTein to create models of the transition of C. japonica from interphase to prophase and study the possible mechanisms as well. They found the holocentromere was made up only of a few, evenly spaced CENH3-positive megabase-sized satellite arrays. Overall, the reason for the convergent evolution of holocentromeres from a monocentromere may stem from multiple factors, but more experiments like the ones presented will surely provide further analysis into this complex and fascinating case of convergent evolution.
Kuo, YT., Câmara, A.S., Schubert, V. et al. Holocentromeres can consist of merely a few megabase-sized satellite arrays. Nat Commun 14, 3502 (2023). https://doi.org/10.1038/s41467-023-38922-7

Tick infestations are a recurring roadblock of human development around the world, with estimated damages in the global economic landscape being as high as 30 billion USD. Specifically, India has long been susceptible to tick-borne diseases, due to multiple species invading the livestock. These regional parasites are major vectors for Crimean–Congo hemorrhagic fever virus (CCHFV), a disease with a devastating case fatality rate of 10–40%. While the main way of combatting the infestation of ticks and their carried disease has always been pesticides, often to an invasive degree of their own, scientists are working diligently for ways to produce a vaccine for this deadly and prevalent outbreak. One such method that has been explored is multi-epitopic peptide vaccines that combat Crimean–Congo hemorrhagic fever virus, specifically through the potential immune stimulatory responses they cause.

Multi-epitopic peptides help boost immune system

LifeTein provided the group with the two designed multi-epitopic peptides, VT1 and VT2. Using the two peptides, the group put them into two working vaccines in an effort to explore how effective they were at fighting back the ticks. With rabbits, they found strong immunity conferred by the vaccine, displayed by quick larval detachment, delayed tick feeding, low engorgement weights, and overall efficacy against both tick larvae and adults. The results show just how effective treatment with the vaccines are against ticks carrying CCHFV, and the compatibility with rabbits is a great starting point.

In an ideal experiment, the group would have tested on cattle, since that is a much more affected group by these ticks. Regardless, the suitability and stability displayed warrants more attention be put into these multi-epitopic peptide vaccines. Their efficacy displayed against infestations as such is sure to save the global economy billions, as well as countless lives. Immunization in this route is surely more appealing than that of constant and overwhelming pesticides being put in place at every conceivable turn. LifeTein is excited to see where else peptide-based vaccines can be implemented and what other unique properties they can bring to the table.

Nandi A, Manisha, Solanki V, Tiwari V, Sajjanar B, Sankar M, Saini M, Shrivastava S, Bhure SK, Ghosh S. Protective Efficacy of Multiple Epitope-Based Vaccine against Hyalomma anatolicum, Vector of Theileria annulata and Crimean–Congo Hemorrhagic Fever Virus. Vaccines. 2023; 11(4):881. https://doi.org/10.3390/vaccines11040881

Cell-penetrating peptides (CPPs) are a longstanding part of the biochemical world, with their potential for delivering bioactive agents into cells, their importance will likely never diminish. However, not many advancements are being made to CPPs, rather scientists and teams alike are always looking to develop more efficient CPPs when possible. This is where a hot topic of recent comes into play, researchers utilized a deep-learning-based CPP prediction method, one they have named AiCPP, to effectively develop novel CPPs while reducing false-positive predictions. Using LifeTein cell-penetrating peptides and machine learning, AiCPP was able to combine data with other lists of CPPs to generate successful new sequences that the team at hand was able to test effectively.

LifeTein CPPs help train AI to generate new sequences

The group utilized a sliding window approach on their wealth of data and included a list of peptides with low similarity to CPPs as a negative to reduce false positives obtained. These techniques helped AiCPP stand out against other machine learning methods before it, and they found AiCPP can further optimize CPP sequences with higher efficacy as well. Valuable information can be gathered by studying the patterns by which this machine learning determines are effective for CPPs.

Though novel, the limitations of utilizing this and other machine-learning methods should be understood as well. Foremost, this and other CPP prediction studies do not answer any important questions about the mechanisms of cell permeation, or how each CPP specifically achieves this. It is also worth noting that the effectiveness of a given CPP is limited to the type of cell is it trying to penetrate, for example, MCF-7 used in the referenced study. Though, these shortcomings are possible to overcome, and in the future, AiCPP may become more advanced and be able to offer even more research, with LifeTein keeping up as well and ready to assist any team that may need their new CPPs developed.

Park H, Park J-H, Kim MS, Cho K, Shin J-M. In Silico Screening and Optimization of Cell-Penetrating Peptides Using Deep Learning Methods. Biomolecules. 2023; 13(3):522. https://doi.org/10.3390/biom13030522